Chlamydia trachomatis is an intracellular pathogen that is responsible for significant human morbidity throughout the world. Under the funded grant linked to this proposal (5 R01 AI039558-07) we have been identifying and testing a number of T cell antigens for their role in protective immunity to C. trachomatis. Through the work described in this Exploratory/Developmental application, we propose to use the published genome sequence and fluorescence in situ hybridization (FISH) technology to identify candidate C. trachomatis T cell antigens based on their expression at the relevant time in the developmental cycle and their expression in the relevant tissues. Little is known about gene expression in these organisms during their developmental cycle because of the difficulty in synchronizing Chlamydia infections. The use of FISH technology circumvents the problem of asynchronous infections because we will be able to analyze the expression of Chlamydia genes at the level of individual bacteria. In the first Specific Aim, we propose to use FISH to explore the developmental cycle of C. trachomatis in cultured cells. Using FISH, we should be able to detect induction or repression of genes that were previously undetectable using other techniques. After optimizing probe design and hybridization conditions, we will use FISH to simultaneously monitor the expression of multiple genes within an organism using probes tagged with different fluorescent dyes. We will then compile a database containing the relative levels of expression of many C. trachomatis genes at various times in the developmental cycle. This information will be applied to the rational identification of candidate T cell antigens based on their expression early in intracellular development, when a T cell response would be most effective. The database will also aid in deciphering the pathways and regulatory networks present at each developmental stage. In the second Specific Aim, we propose to use FISH to study Chlamydia gene expression in genital tissues during murine infection. In particular, we will look at the temporal and spatial differences in Chlamydia gene expression in these tissues. We also propose to analyze changes in Chlamydia gene expression under various host immunological or chemotherapeutic pressures. These data will also allow for the identification of candidate antigens expressed in appropriate tissue types at times when T cell recognition would be most effective.